Cloning And Characterization Analysis On Non-structural Protein Gene And UTR Of Transmissible Gastroenteritis Virus Of Swine

Transmissible gastroenteritis virus (TGEV) is causing fatal diarrhoea in newborn piglets. In recent years, the research work on TGEV was focused on its structural proteins and there had no report on the non-structural protein gene and non-encoding regions in China. Until now, the only published complete sequence of TGEV was the one of the Purdue strain. In order to study the characterization and function of TGEV, thirteen pair of primers were designed according to the nucleotide sequence of TGE Purdue strain to amplify the ORF1(polymerase gene), ORF3, ORF7 and untranslated region (UTR) of the TS strain of TGEV.The fragments obtained were sequenced and analysed and the results are as follows:1) We obtained the complete sequence of TGEV TS Strain and submitted it to the Genbank, it is the first complete sequence of TGEV in China that was submitted to the GenBank.2) The polymerase gene was obtained by RT-PCR using specific primers and it was found to be 20054 bp in length and contained two ORFs . The percentage of identity at the nucleotide level of ORF1 between TGEV TS and TGEV Pur46-MAD was found to be 98.8%, The deduced aa sequence identity between their corresponding predicted proteins was 99.0%. The deduced amino acid sequence identity of ORF1 between TS and FIPV, PEDV, HCV299E, SARS was found to be 87%, 57%, 57% and 45%, respectively. Sequence analysis also revealed that RdRp, Hel, UP2 and UP3 region of Polymerase gene were conserved among the coronavirus, there is a ribosomal slippage site, UUUAAAC, and three stem-loop structures in the ORF1a and ORF1b overlapping region.3) ORF3 and ORF7 of TGEV strain TS were obtained by RT-PCR and were found to be 1487 bp and 516 bp in length, respectively. The percentage of identity at the nucleotide level of ORF3 between TGEV TS and CHV, Purdue was found to be 99.3% and 97.9%, respectively. At the amino acid level, the sequence identity for ORF3a was 97.3% and 87.8% and for ORF3b it was 98.4% and 96.3%, respectively. The results indicated that there is no deletion in the TGEV strain TS ORF7 and it is highly conserved. The region from nucleotide 192 to the TAA stop codon in ORF3a is considered as the highly variable region of TGEV, and our results revealed there were differences in the number of predicted amino acid and the RNA-binding site between the TGEV and PRCV.4) A fragment of 319bp and a fragment of 280bp that were considered to be 5'UTR and 3'UTR of the genome DNA of TGEV strain TS were obtained. The region from nucleotides 117 to 129 in the 5'UTR encoded a ORF with four amino acid . 5'UTR and 3'UTR were found to be conserved among different TGEV strains and they have complex secondary structure.5) Amino acid sequence comparison of ORF3 and ORF7 between different strains of TGEV indicates that TGEV strain TS has a high degree of homology with the Miller strain. It made us hypothesize that TGEV strain TS may have originated from the TGEV strain Miller.